SIGMAR1 Regulates Membrane Electrical Activity in …cancerres.aacrjournals.org/content/canres/76/3/607.full.pdf · SIGMAR1 Regulates Membrane Electrical Activity in Response to Extracellular

Molecular and Cellular Pathobiology

SIGMAR1 Regulates Membrane Electrical Activityin Response to Extracellular Matrix Stimulationto Drive Cancer Cell InvasivenessDavid Crott�es1,2,3,4, Raphael Rapetti-Mauss1,2,3, Francisca Alcaraz-Perez5,6,M�elanie Tichet7, Giuseppina Gariano8, Sonia Martial1,2,3, H�el�ene Guizouarn1,2,3,Bernard Pellissier1,2,3, Agn�es Loubat1,2,3, Alexandra Popa9, Agn�es Paquet9,Marco Presta8, Sophie Tartare-Deckert7, Maria Luisa Cayuela5,6, Patrick Martin1,2,3,Franck Borgese1,2,3, and Olivier Soriani1,2,3

Abstract

The sigma1 receptor (Sig1R) is a stress-activated chaperone thatregulates ion channels and is associated with pathologic condi-tions, such as stroke, neurodegenerative diseases, and addiction.Aberrant expression levels of ion channels and Sig1R have beendetected in tumors and cancer cells, such asmyeloid leukemia andcolorectal cancer, but the link between ion channel regulation andSig1R overexpression during malignancy has not been estab-lished. In this study, we found that Sig1R dynamically controlsthe membrane expression of the human voltage-dependent Kþ

channel human ether-�a-go-go-related gene (hERG) in myeloidleukemia and colorectal cancer cell lines. Sig1R promoted theformation of hERG/b1-integrin signaling complexes upon extra-

cellular matrix stimulation, triggering the activation of the PI3K/AKT pathway. Consequently, the presence of Sig1R in cancer cellsincreased motility and VEGF secretion. In vivo, Sig1R expressionenhanced the aggressiveness of tumor cells by potentiating inva-sion and angiogenesis, leading to poor survival. Collectively, ourfindings highlight a novel function for Sig1R in mediating cross-talk between cancer cells and their microenvironment, thus driv-ing oncogenesis by shaping cellular electrical activity in responseto extracellular signals. Given the involvement of ion channels inpromoting several hallmarks of cancer, our study also offers apotential strategy to therapeutically target ion channel functionthrough Sig1R inhibition. Cancer Res; 76(3); 607–18. �2015 AACR.

IntroductionDuring the past 10 years, the role of ion channels in cancer has

arisen. The aberrant expression of ion channels, often absent fromhealthy tissue, participates to hallmarks of cancer by tuningmembrane potential, calciumhomeostasis, or signaling pathways(1–5). For example, KCNH2 (human ether-�a-go-go-related gene,hERG), a voltage-dependent Kþ channel mainly involved incardiac activity (6), has been characterized as a biomarker ofmany solid tumors (colorectal cancer, glioblastoma, head and

neck cancers; refs. 7, 8) and acute or chronic leukemias [acutemyelogenous leukemia (AML), acute lymphoblastic leukemia(ALL), and chronic myelogenous leukemia (CML), respectively;refs. 9, 10]. hERGexpression is sufficient to induce transformationof fibroblasts (11), and in cancer cells, it forms membrane plat-forms with receptors of the tumor cell microenvironment, such asintegrins [adhesion receptors of the extracellular matrix (ECM)],that deeply influences signaling pathways controlling in turncancer cell spreading (12).

Targeting proteins regulating ion channel activity in cancer cellsappears as a promising way to control cancer cell behavior. Ourinterest focused on the sigma 1 receptor (Sig1R), a 25-kDachaperone protein mainly expressed in the brain and residing atthe endoplasmic reticulum (ER)–mitochondria interface (mito-chondria ER-associated membrane, MAM; refs. 13–16). Sig1Rfunctionally interacts with ion channels from various families,including voltage-dependent channels (17–19), volume-regulat-ed chloride channels, acid-sensing ion channels (20), and N-methyl-D-aspartic acid receptor (NMDA; refs. 21, 22). Therefore,Sig1R has been associated to many pathophysiologic contexts,including stroke, neurodegenerative diseases, pain, or cocaineaddiction (15). Interestingly, Sig1R is overexpressed in cancercell lines (23), andwehave shown that it functions as a chaperoneenhancing maturation and membrane expression of hERG by adirect physical interaction (24, 25).

To characterize Sig1R function in cancer cell physiology, weinvestigated its involvement in signalling events betweenhERG and the ECM in myeloid leukemia and colorectal cancercells.

1Universit�e Nice Sophia Antipolis, iBV, Nice, France. 2CNRS, iBV,UMR7277, Nice, France. 3INSERM U1091, Nice, France. 4Departmentof Physiology, University of California, San Francisco, San Francisco,California. 5Telomerase, Aging and Cancer Group, Research Unit,Department of Surgery, CIBERehd, University Hospital "Virgen de laArrixaca", Murcia, Spain. 6Instituto Murciano de Investigaci�on Biosa-nitaria (IMIB), Murcia, Spain. 7Universit�e Nice Sophia Antipolis, C3M,Inserm U1065, Nice, France. 8Unit of Oncology and ExperimentalImmunology, Department of Molecular and Translational Medicine,University of Brescia, Brescia, Italy. 9Universit�e Nice Sophia Antipolis,IPMC, CNRS UMR7275, Sophia Antipolis, France.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

D. Crott�es and R. Rapetti-Mauss contributed equally to this article.

Corresponding Author: Olivier Soriani, UNS-CNRS, 28 Av de Valrose, 06000Nice, France. Phone: 00 33 (0)4 92 07 65 53; Fax: 00 33 (0)4 92 07 68 34; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-15-1465

�2015 American Association for Cancer Research.

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Herein, we show an overexpression of Sig1R in human AML,CML, and colorectal cancer.We also identify this protein as a keyregulator in the ECM-induced formation of hERG–b1-integrincomplex in cancers cells, stimulating motility and VEGF secre-tion. At the molecular level, Sig1R increases hERG membranedensity, promoting the formation of hERG/Sig1R/b1-integrincomplex and activating the AKT signalling pathway. In vivo,Sig1R silencing in leukemia or colorectal cancer cells results inreduced invasion, angiogenesis, and extravasation. Last, Sig1Rexpression is associated to a poorer survival in AML patients.

Materials and MethodsChemicals, antibodies, reagents, and human cancer dataset

analysis are described in Supplementary Methods.

Cell cultureK562,HCT-116, HEK-293, andNIH-3T3 cell lines are kind gifts

form S. Brown (Queen's Medical Research Institute, Edinburgh,Scotland), A.O. Hueber and G. Sandoz (iBV, University of NiceSophia Antipolis, France), and I. Mus-Vetau (IBDC, University ofNice Sophia Antipolis, France), respectively, and were cultured aspreviously described (24). All cells lines were certified by shorttandem repeat DNA typing (DSMZ).

shRNA transductionControl or Sig1R-silenced cells were established as previously

described (24). Briefly, lentiviral particles were obtained fromSigma "MISSION shRNA Lentiviral transduction particles" (Sig-ma-Aldrich). At day 1, K562 were plated in a 6-wells plate at adensity of 20,000 cells/well in complete medium. At day 2,medium was removed and cells were incubated in completemedium containing 8 mg/mL of hexadimethrin bromid (Sigma-Aldrich) and transduced at an MOI of 5. Clones SHC002V (non-target shRNA, shRD), SHVRC-TRCN0000061011 (shSig1R), andSHVRC-TRCN0000061010 (shSig1R(2)) were used for transduc-tion. At day 3, a new transduction round was applied. At day 6,puromycin (0.5 mg/mL) was added in fresh medium to startselection of transduced cells.

Generation of fibroblast-derived matricesThe protocol used was the same as described by Beacham and

colleagues (26).

Patch-clamp experimentsCells were prepared as previously described (24, 27).

Immunoprecipitation and Western blot experimentshERG currents were recorded as previously described (24).

Flow cytometry and flow cytometry–based F€orster resonanceenergy transfer assay

K562 cells were fixed in PBS 1� PFA 4% for 10 minutes andwere successively incubated for 30minutes at 4�C in a solution ofPBS/FBS 3%/EDTA 2mmol/L containing the primary antibody atthe appropriate dilution. For unconjugated-primary antibodies,cells were washed and incubated in a solution containing thefluorophore-conjugated secondary antibodies. Cells were ana-lyzed with BD LSR Fortessa and FACS Diva software (BectonDickinson) on the basis of 10,000 events. The methodologyof flow cytometry–based F€orster resonance energy transfer (FRET)assaywas previously described (28–30). Briefly, the donor proteinwas stained with Alexafluor 488–conjugated antibody, and theacceptor protein was stained with Alexafluor 594–conjugatedantibody. Tomeasure Alexafluor 488 and FRET signals, cells wereexcited with a 488-nm laser, and fluorescence was collectedrespectively with an LP mirror of 505 nm and a BP filter of530/30 nm, or with an LP mirror of 600 nm and a BP filter of610/20 nm. Tomeasure AlexaFluor 594 signals, cells were excitedwith a 588-nm laser, and fluorescence was collected with an LPmirror of 600 nm and a BP filter of 610/20 nm.

Immunofluorescence and confocal analysisFor colocalization experiments, cells were plated on uncoated

or fibroblast-derived matrices (FDM)–coated glasses. After 30minutes, cells were fixed with PBS 1� PFA 4% and subsequentlyincubated during 1 hour at 4�C with mouse anti–b1-integrin andgoat Alexafluor 594–conjugated anti-mouse. For actin cytoskel-eton visualization, cells were plated for 3 hours on differentsubstrates before fixation, then permeabilizedwith PBS 1� TritonX100 0.1%, blocked with PBS BSA 5%, and incubated with FITC–phalloidin 1�. Cell nuclei were stained with Hoescht 33342, andglasses were mounted with mounting medium (Fluka) ontocoverslips. Acquisitions were performed on a confocal spinningdisk microscope (Olympus) equipped with an ANDOR cameraand a 100� oil objective.

Figure 1.Sig1R is overexpressed in myeloid leukemias and colorectal cancers (CRC). Intensity of Sig1R mRNA expression in human CML, AML, and colorectal cancer patients.mRNA expression values in cancer patients (black boxes) are compared with corresponding normal tissues (white boxes).

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Figure 2.Sig1R contributes to ECM-induced stimulation of hERG Kþ current in CML and colorectal cancer cell lines. A, left, representative hERG tail currents recordedin K562 cells plated on uncoated or FDM-coated dishes in the presence or absence of b1-integrin–blocking antibody (1 mg/mL). hERG currents were measured30 minutes after cell seeding at �120 mV during 5 seconds after a 4-second prepulse at þ60 mV to fully activate hERG. Right, corresponding current amplitudehistogram. Values are mean � SEM (n ¼ 6–16 cells; �� , P < 0.01; �� , P < 0.001). B, left, hERG tail currents recorded in HCT-116 cells plated on uncoated orfibronectin-coated dishes. Middle, Western blots showing the Sig1R extinction in shSig1R HCT-116 when compared with control cells (shRD). Right, correspondinghistogram. Values are mean� SEM (n¼ 10–14 cells; �� , P < 0.02; �� , P < 0.001). FN, fibronectin. C, left, hERG channel density at the plasma membrane measured byflowcytometry. Right, corresponding histogram showing hERG surface expression in each condition. Ratio valueswere calculated by dividing theMFI of hERGby theMFI of the isotype. Values are mean � SEM of six to eight independent experiments (� , P < 0.05; �� , P < 0.01). N.S., nonsignificant.

Sig1R Stimulates Cancer Cell Invasiveness

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Figure 3.Sig1R promotes the formation of FDM-induced B1-integrin/hERG complexes. A, representative images showing membrane distribution of GFP-tagged proteins(green) and b1-integrin (red) staining and colocalization areas (yellow). (Continued on the following page.)

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DuoLink in situ proximity ligation assayTo detect the interaction between hERG and b1-integrin, we

utilized the DuoLink in situ Proximity Ligation Assay (PLA;Olink Bioscience) according to the manufacturer's protocol.HCT-116 cells were seeded on poly-Lysine or fibronectin(40 mg/mL)-coated microscope slides for 2 hours. Cells wereimmunolabeled with primary antibodies: hERG1 extracellular(Alomone; 1:100) and anti-CD29 TS2/16 (BioLegend; 1:100)for 1 hour at 37�C. The secondary antibodies with attached PLAprobes were supplied in the Duolink Kit. Images were collectedon an inverted ZEISS Axio Observer Z1 microscope (Zeiss)

equipped with a Zeiss 63X Plan-Apo DICIII oil NA 1.4 objectivelens. Images were acquired with a monochrome EMCCDiXONþ897 camera (Andor) controlled with the Metamorph7.8 software (Molecular Devices). A fluorescence signal indi-cates that two proteins are separated by <40 nm.

Analysis of cell spreadingCells were stained with carboxyfluorescein succinimidyl ester

(CFSE; 5 mmol/L) the day prior the experiment. At different timepoints after spreading, cells were fixed with PBS 1� PFA 4%, andslides were mounted with mounting medium (Fluka) onto cover

(Continued.) Colocalization between GFP-tagged protein and b1-integrin is expressed as the increase of the Pearson coefficient in FDM when compared withuncoated condition (right; n ¼ 12 to 20 cells from two independent experiments; �� , P < 0.001). B, flow cytometry–based FRET assay for the identificationof hERG/b1-integrin complexes at the plasma membrane. C, left, representative FACS-contour plots showing the amount of FRET-positive cells in total shRD orshSig1R cells populations, plated for 30 minutes on uncoated or FDM-coated dishes. The left column shows FRET signals in cells stained with either anti hERG orb1-integrin antibodies. Right, corresponding histograms representing the mean (�SEM) of FRET-positive cells in each condition (six to seven independentexperiments; � , P < 0.05). D, in situ proximity ligation assay quantifying the number of complexes between hERG and b1-integrin in shRD or shSig1R HCT-116 cellsseeded on polylysine (PL) or fibronectin (FN). Left, upper micrographs show protein interaction areas (yellow dots) and cell nucleus stained with DAPI (blue); lowermicrographs show corresponding transmission images. Right, corresponding quantification of dot/cell density. Results are representative of three independentexperiments. Twenty fields per condition were analyzed in each experiment (�� , P < 0.01; Student t test). N.S., nonsignificant.

Figure 4.Sig1R silencing inhibits FDM-triggered AKT signaling pathway. A, total protein extracts from shRD, shSig1R, or shhERG K562 cells plated for 30minutes on uncoatedor FDM-coateddishes in the presenceor absence of hERG inhibitor, E-4031 (50mmol/L),were separatedonSDS-PAGEand immunoblottedwith antiphosphorylated-AKT (Ser 473), AKT, and Sig1R antibodies (representative immunoblots from three to six independent experiments). B, corresponding histogram showing thephosphorylation of AKT ratio for each condition (phosphorylated vs. total AKT; values are mean� SEM; � , P < 0.05; �� , P < 0.01). C, same experiment with shRD andshSig1R HCT-116 cells plated on uncoated or FDM-coated dishes. D, corresponding histograms (values are mean � SEM; �� , P < 0.01, n ¼ 3). N.S., nonsignificant.






Figure 5.FDM stimulates cell migration and actin organization in an Sig1R manner. A, representative 3-hour time lapse tracking plots from shRD, shSig1R K562, orshhERG cells plated on either uncoated or FDM-coated dishes in the absence (FDM) or in the presence of E-4031 (50 mmol/L) or LY 294002 (10 mmol/L).B, corresponding histograms quantifying motility parameters. Left, total length traveled per cell. (Continued on the following page.)

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glasses. Acquisitions were performed on a confocal spinning diskmicroscope (Olympus) at 60� objective. Circularity coefficientswere measured with Image J software (NIH).

Time-lapse motility assayCells were stained with 5 mmol/L CFSE (Invitrogen) and plated

on a 6-well plate. One h later, cell motility was acquired using aNikon Ti Eclipse microscope for 3 hours using thermostatedchamber and CO2 pressure. At least 130 individual cells percondition andper experimentwere trackedwith the Fiji/MTracks2software (NIH).

Transendothelial migrationHuman umbilical vein endothelial cell (HUVEC) primary

endothelial cells were grown in EGM-2 and were cultured onthe upper membrane surface of a Boyden chamber overnight togenerate a confluent monolayer. K562 cells were seeded intothe HUVEC-coated Boyden chamber membrane. Cells wereseeded into noncoated plates as a suspension control. Cellswere incubated at 37�C with 5% CO2 during 24 hours. Then,migrated cells in the lower chamber were harvested andcounted in triplicate by using BD LSR Fortessa (High Through-put System).

VEGF secretion assayK562 cells were seeded onto fibronectin-coated 12-well plates

(3 � 105 cells per well), in RPMI medium depleted of FBS, andincubated for 48 hours. VEGF concentration was measured in cellculture supernatants using a standard ELISA protocol (humanVEGF-A ELISA kit; Thermo Scientific). VEGF concentrations werenormalized to the corresponding cell number. Each experimentwas performed in triplicate.

Zebrafish invasion assaysZebrafish (Danio rerio) wild-type AB strain, from the Zebrafish

International Resource Centre, was maintained in recirculatingtanks according to standard procedures (The zebrafish handbook: alaboratory use of zebrafish, Brachydanio rerio). The experiments per-formed comply with the Guidelines of the European Union Coun-cil for animal experimentation (86/609/EU) andwere approved bythe Bioethical Committee of the University Hospital Virgen de laArrixaca. K562were stainedwith the vital cell tracker redfluorescentCM-Dil (Vibrant; Invitrogen), centrifuged, resuspended in 67% ofDPBS, 5 FCS, 0.05% Phenol Red, and injected into the yolk sac ofdechorionated zebrafish embryos using a method described pre-viously (31). Fishes with fluorescent cells outside the implantationarea at 2 hours were excluded from further analysis. All other fisheswere incubated at 35�C for 48 hours and analyzed with a SteReoLumar V12 stereomicroscope with an AxioCamMR5 camera (CarlZeiss). Evaluation criteria for invasion were that at least three cellshad to be identified outside of the yolk.

Tumor cell zebrafish embryo angiogenic assayExperiments were performed as already described with minor

modifications (see Supplementary Methods; ref. 32).

Lung extravasation assayK562 transfected with shRD or shSig1R were fluorescently

labeled with CellTracker Green. A total of 107 cells (Sig1R-depleted or control cells) were injected into the tail vein ofHairless NOD.SCID (Harlan Laboratories) mice sacrificed 30minutes or 24 hours later. Lungs were harvested for analysis witha 5� Zeiss Inverted scope. Lungs harvested at 24 hours wereperfused to eliminate cells remaining in blood system and allowthe detection of evading cells.

Statistical analysisUnless otherwise stated, statistical differences between con-

trol and shSig1R cell populations were assayed by the Mann–Whitney test. In all cases, a P value less than 0.05 was consid-ered as significant.

ResultsSig1R is overexpressed in human cancers

Using the Oncomine database (33), we found that Sig1R gene(SIGMAR1) was significantly overexpressed in CML, AML, andcolorectal cancer patients when compared with correspondingnormal tissues (Fig. 1). Interestingly, in these three cancer types,tumor cell aggressiveness is associated with hERG expression(10, 34). We then investigated the consequences of Sig1R expres-sion on hERG-related phenotype in CML and colorectal cancercells.

FDM promotes hERG current density through a rapid Sig1R-dependent recruitment of channels at the surface

In leukemic and colorectal cancer cells, hERG current canbe stimulated by b1 subunit of integrin heterodimers, which arethemselves activated by ECM components, such as fibronectin(7, 9, 10). To assess whether Sig1R participates to the dynamicregulation of hERG channel in response to ECM stimulation,patch-clamp experiments were performed. hERG currents in con-trol (shRD) and Sig1R-silenced (shSig1R and shSig1R(2); Sup-plementary Fig. S1A) K562 CML cells were compared (Fig. 2;ref. 24). Currents were recorded 15 minutes to 3 hours after cellseeding either on plastic dishes or on dishes coated with fibro-nectin-rich fibroblast-derived 3D matrix, which mimics in vivomesenchymal matrices (26, 35) with a composition similar tothose observed in the bone marrow (36). In control cells platedon uncoated dishes, tail current density peaked at 13.52 � 2.02pA/pF. Seeding cells on FDM increased hERG current densityby around 96% (Fig. 2A). The increased current was maintainedfor at least 3 hours (Supplementary Fig. S1B). As expected (10),fibronectin coating similarly increased hERG current density(Supplementary Fig. S1C). Using the same protocol, Sig1R-silenced cells did not significantly change current density in

(Continued.) Right, Euclidian distance traveled per cell. Values are mean � SEM recapitulating the tracking of 202 to 410 cells (from two or three independentexperiments; t test � ,P <0.05; �� ,P <0.01; �� ,P <0.001). C, shRD and shSig1RK562 cells plated on either uncoated or FDM-coated glasses in the absence or presenceof E-4031 (50 mmol/L) or LY294002 (10 mmol/L). After 3 hours, cells were fixed, permeabilized, and stained for actin using FITC-conjugated phalloidin.Representative images showing spike actin organization (green labeling) in each condition. Right, corresponding histogram representing themean (�SEM) of actinspikes per cells in each condition (n ¼ 15 to 40 cells from two independent experiments; �� , P < 0.01; �� , P < 0.001). N.S., nonsignificant.






Figure 6.Sig1R drives cancer cell invasiveness potency in vivo and reduces overall survival. A, Sig1R-dependent in vivo invasion of K562 or HCT-116 cells from the yolk sac ofzebrafish embryos. Top, experimental protocol. Left, representative images of zebrafish embryos negative or positive for K562 cell invasion. Right, histogramsshowing the percentage of positive zebrafish embryos for K562 and HCT-116 cells expressing (shRD) or not (shSig1R) Sig1R. (Continued on the following page.)

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uncoated condition, but FDM-induced stimulationwas complete-ly abolished (Fig. 2A). FDM-induced current stimulation wasabrogated by a blocking antibody directed against the b1-integrinsubunit that had no effect in Sig1R-silenced cells, suggestingthat hERG regulation by the b1-integrin requires Sig1R expression(Fig. 2A). Interestingly, fibronectin-induced stimulation of hERGcurrent density was also reduced by Sig1R inhibition in the HCT-116 colorectal cancer cells line (Fig. 2B).We next checked channeldensity at the plasma membrane of K562 cells by flow cytometryusing an antibody directed against an extracellular loop of thechannel. FDM significantly enhanced hERG surface expression incontrol cells, but had no effect when Sig1R expression wasrepressed (Fig. 2C).Wewondered whether FDM could transientlystimulate hERG maturation and whether this effect could beabrogated by Sig1R suppression. Western blot experiments,performed on K562 cells plated for 30 minutes on either plasticor FDM, revealed that FDM-induced stimulation of the currentwas not due to a transient stimulation of hERG maturation(Supplementary Fig. S1C–S1E). Altogether, these results showthat FDM induces a rapid recruitment of hERG channels to theplasma membrane in a Sig1R-dependent manner.

Sig1R promotes the formation of FDM-induced b1-integrin/hERG complexes

In leukemic and colorectal cancer cells, hERG current stimula-tion by ECM is accompanied by hERG binding to b1-integrinsubunits (7, 10). The involvement of Sig1R in the formation ofsuch channel signaling macrocomplexes was assessed. In K562cells transiently transfected with GFP-hERG and stained for extra-cellular b1-integrin, confocal microscopy experiments showedthat FDM induced at 30 minutes a 3.5-fold increase in hERGcolocalization with b1-integrin subunit at the plasma membrane(Fig. 3A). This effect was abolished by Sig1R silencing (Fig. 3A). InK562 cells transiently transfected with GFP-Sig1R, FDM alsoprovoked a 2.5-fold increase in b1-integrin/Sig1R colocalization(Fig. 3A). In parallel, coimmunoprecipitation experiments weredone in three previously established HEK293T cell lines stablyexpressing cmyc-Sig1R alone, hERG1a with c-mycSig1R, andhERG1a with an shRNA targeting Sig1R (24). The b1-integrinsubunit constitutively expressed in HEK293 cells was immuno-precipitated, andwe checked for hERGand cmycSig1Rpresence inthe precipitate. Both proteins were coimmunoprecipitated withb1-integrin. In the absence of Sig1R, only low levels of hERGcouldbe detected after b1-integrin immunoprecipitation (Supplemen-tary Fig. S2A). These data strongly suggest that Sig1R is necessaryto promote the hERG/b1-integrin interaction and that Sig1R itselfis included within the complex. To further confirm this mecha-nism, we performed a flow cytometry–based FRET assay onnonpermeabilized cells to detect hERG/b1-integrin physical inter-action atK562 cell surface (28–30). To assess a correct FRET signal,K562 cells stained with only one of both fluorochromes wereused as control to determine the FRET-positive gate (FRETþ). b1-

integrin andhERGwere respectively stainedwithAlexaFluor 488–conjugated and AlexaFluor 594–conjugated antibodies and wereused as donor and acceptor (Fig. 3B). FDM induced a significantincrease in FRET-positive cells, which was abolished in Sig1R-silenced K562 cells (Fig. 3C). Moreover, FDM or Sig1R silencinghad no effect on b1-integrin surface expression (SupplementaryFig. S2B). We next wondered whether the same mechanismoccurred in adherent cancer cells by PLA in control (shRD)and Sig1R-silenced colorectal cancer HCT-116 cells. fibronectincoating increased the number of b1-integrin/hERG complexesin control cells, but the number of complexes was significantlyreduced in Sig1R-silenced cells (Fig. 3D; Supplementary Fig. S3).Altogether, these data indicate that the formation of integrin/hERGchannelmembrane complexes in response to ECM is drivenby Sig1R in leukemic and colorectal cancer cells.

Sig1R silencing inhibits FDM-triggered AKT signaling pathwayThe formation of hERG/b1-integrin platforms in response to

ECM is known to trigger PI3K/AKT signalling pathways in acuteleukemias and colon cancer cells (10, 34). Therefore, we won-dered if Sig1R inhibition could alter AKT phosphorylation in thecontext of K562 andHCT-116 cell interaction with FDM.Westernblot experiments showed that the relative proportion of phos-phorylated-AKT increased for FDM-challenged control cells(Fig. 4). As expected, inhibiting hERG with E-4031 (a specificblocker of hERG) or by shRNA abolished FDM-dependent AKTactivation (Fig. 4A and B). In the same manner, Sig1R silencingsuppressed FDM-induced AKT phosphorylation in both cell types(Fig. 4). E-4031 had no effect on FDM-stimulated Sig1R-silencedcells, pointing out the absence of any additive effect betweenSig1R and hERG (Fig. 4). These results indicate that the pivotalrole of Sig1R in b1-integrin/hERG channel complex formation atthe plasma membrane is mirrored at the intracellular level by theactivation of the AKT signaling pathway.

FDM stimulates cell migration and actin organization in aSig1R-dependent manner

In many cancers, upregulation of AKT signaling is related tohighly migrating cell phenotype (10, 37). Sig1R regulation of thehERG/b1-integrin/AKT pathway in response to FDM stimulationsuggests that Sig1R inhibition could alter K562 cell migrationpotency. Time-lapse recording of shRD and shSig1R K562 cellsplated on either uncoated or FDM-coated dishes for 3 hours wasdone (Fig. 5A and B). Cell motility was evaluated by measuringthe traveled length and the Euclidian distance traveled during thelap of acquisition time. In control cells, FDM promoted cellmotility by increasing the traveled length and distance (Fig.5B). The effects of FDM on both parameters were lowered byhERG or PI3K inhibition (Fig. 5A and B), indicating the contri-bution of hERG and PI3K/AKT pathways in FDM-dependent cellmigration stimulation. Silencing Sig1R expression significantlyreduced FDM-induced motility and E-4031 or LY294002 had

(Continued.) The total of embryos is indicated inside the figure for each condition (values are mean � SEM of three to five independent experiments; �, P < 0.05;�� , P < 0.001). B, xenograft in zebrafish for angiogenesis assay. Top, experimental protocol. Left, representative images of a zebrafish negative and positive for K562-induced angiogenesis. Asterisks indicate the injection area. Right, histograms representing the percentage of positive and negative zebrafish embryos from a totalnumber stated in the figure for each condition (t test; � , P < 0.05). C, pulmonary extravasation of K562 cells injected in NOD-SCID hairless mice tail vein. Top,experimental protocol. Bottom, representative images showing localizationof shRDor shSig1RK562 cells in lung30minutes and24hoursafter injection; correspondinghistogram of the number of K562 cells per field in lung (values are mean� SEM from three independent experiments; � , P < 0.05). D, Kaplan–Meier analysis of overallsurvival in AML patients. Patients from the TCGA AML cohort were dichotomized into SigR1-high (red) and SigR1-low (black) groups using the median SigR1 value ascutpoint. A significant difference in overall survival was observed between these two groups (P ¼ 0.014).






weaker or nonsignificant effects on traveled distance or length(Fig. 5A and B). Interestingly, HCT-116 colorectal cancer cellmotility was inhibited by Sig1R silencing and by E-4031 in anonadditive manner (Supplementary Fig. S4).

TheAKTpathway is linked to cellmigration through the controlof cell shape and actin cytoskeleton remodeling (38). To addresswhether Sig1R inhibition would impact actin reorganizationfollowing seeding on FDM, we imaged polymerized actin byconfocal microscopy in K562 cells. After 3 hours, control cellsseeded on uncoated dishes showed a low number of individualactin spikes at cell periphery. By contrast, FDM coating induced a3-fold increase in spike number, an effect reversed by E-4031 orLY294002 treatment. In shSig1R K562 cells, FDM failed to sig-nificantly increase the number of spikes when compared withuncoated condition. In addition, neither E-4031 nor LY294002had any effect on Sig1R-silenced cells (Fig. 5C).Wenextwonderedwhether the apparition of actin spikes was accompanied by anFDM-dependent alteration in cell morphology. K562 cells wereseeded on either uncoated or FDM-coated dishes, and circularitywas quantified using a coefficient varying between 1 (fully round,FR) and 0 (not circular, NC). Cells were then sorted according to acircularity threshold of 0.5 (Supplementary Fig. S5A shows rep-resentative images of K562 cell diversity ofmorphologies on FDMafter 3 hours). FDM increased the proportion of NC cells as afunction of time. This effect was completely abolished in shSig1Rcells (Supplementary Fig. S5B). We next assayed the dependenceof this parameter on hERG and AKT activities. In control cells,FDM increased the proportion of NC cells when compared withuncoated conditions (Supplementary Fig. S5B and S5C). Theeffect of FDM on cell shape was diminished in the presence ofeither hERG (E4031) or AKT (LY294002) inhibitors (Supplemen-tary Fig. S5C). By contrast, FDM only weakly increased theproportion of NC cells in Sig1R-silenced K562. In the latter case,E-4031 andLY294002hadno significant effects onSig1R-silencedcells plated on FDM when compared with controls cells (Supple-mentary Fig. S5C). Taken together, these results suggest that Sig1Rdrives FDM-inducedmorphologic changes through its interactionwith hERG and PI3K/AKT.

Sig1R promotes cell invasiveness potency in vivo and isassociated to poor survival prognosis in human AML

We next studied the consequences of Sig1R downregulation invivo. Tumor xenotransplantation in zebrafish embryo assay wasused (Fig. 6A; ref. 39). Silencing Sig1R expression reduced K562and HCT-116 cell invasive potency in zebrafish embryos whencompared with control shRNA (Fig. 6A), confirming the role ofSig1R on cancer cell invasion in vivo.

hERG/b1-integrin macrocomplexes are also known to pro-mote migration and invasion by stimulating VEGF secretionand further autocrine activation of Flt-1 receptor (10). More-over, in CML and colorectal cancer, VEGF secretion is regulatedthrough the PI3K/AKT signaling pathway (7, 40). We thusasked if Sig1R plays a role in tumor-induced angiogenesis. Weobserved that in vitro, Sig1R expression was strongly associatedto VEGF secretion (Supplementary Fig. S5E). The application ofE-4031 inhibited VEGF secretion in control K562 and HCT-116cells and not in Sig1R-silenced cells, suggesting that Sig1Rpromoted VEGF secretion through the regulation of hERGchannel activity in both CML and colorectal cancer. Interest-ingly, in vivo, using a zebrafish embryo–based model of tumor-induced angiogenesis (32), we observed that the silencing of

Sig1R reduced the ability of K562 cells to induce neoangiogen-esis by about 50% (Fig. 6B).

To further explore the requirement of Sig1R for tumor cellinvasiveness, we investigated the effect of Sig1R in the extravasa-tion step of metastasis. We first observed in vitro that the silencingof Sig1R reduced the ability of K562 cells to transmigrate throughan endothelial HUVECmonolayer by about 60%, as assayed in aBoyden chamber (Supplementary Fig. S5F). Accordingly, using anin vivomodel for tumor cell extravasation in NOD-SCID Hairlessmice (41),we observed that Sig1R silencing decreased the numberof K562 cell evading from the blood compartment into lungtissues by 80% when compared with control cells (Fig. 6C).Altogether, our results suggest a functional requirement ofSig1R in tumor cell invasion, neoangiogenesis, and extravasationin vitro and in vivo. Finally, we explored the prognosis value ofSig1R mRNA expression in AML patients using The CancerGenome Atlas (TCGA) AML dataset (42). In this cohort (N ¼173), the patients with higher Sig1R mRNA expression levelshowed shorter survival time when compared with the patientswith lower Sig1RmRNA levels (HR, 1.6; 95% confidence interval,1.1–2.4; log-rank P ¼ 0.014; Fig. 6D).

DiscussionOur study unveils a new function for the Sig1R chaperone in

oncogenesis. We demonstrate that Sig1R drives cancer cell behav-ior by shaping membrane electrical properties in response to theECM microenvironment.

ECM induced a rapid increase in hERG current density in CMLand colorectal cancer cells, which is consistent with previousstudies showing that hERG can be stimulated by fibronectin, amajor component of ECM (43). Using flow cytometry and con-focal microscopy, we reveal that this effect is likely the conse-quence of an increase in the number of channels at the plasmamembrane. hERG recruitment following cell contact with ECMrequires the presence of Sig1R that behaves as a chaperone drivinghERG to the plasma membrane. In agreement with this hypoth-esis, we and others have shown that Sig1R directly binds withhERG and other ion channels, promoting their activity and theirmembrane expression (15, 19, 25). In the context of ECM stim-ulation, hERG current was increased without modifying channelmaturation ratio (Supplementary Fig. S2; ref. 24), suggesting thatECM triggers the trafficking of available mature hERG channelslocated at the vicinity of the plasma membrane in an Sig1R-dependent manner.

ECM promotes a rapid colocalization of the b1-integrin sub-unit with both hERG and Sig1R at the plasma membrane. Inaddition, Sig1R inhibition reduced hERG/b1-integrin subunitphysical interaction following cell contact with ECM as revealedby FRET and PLA assays in CML and colorectal cancer cells,respectively. These results demonstrate that Sig1R is required forthe dynamic formation of hERG/b1-integrin subunit complex inresponse to a physiologic stimulation of cancer cells by ECM.Several reports have demonstrated that hERG/integrin interactionis a prerequisite for the activation of signaling pathways triggeredby cell contact with ECM (44). By promoting hERG/b1-integrinsubunit interaction, it can be proposed that Sig1R is a keyregulator of the dialogue between cancer cells and their micro-environment. This hypothesis is strengthened at the cellular levelby the fact that Sig1R silencing impaired AKT phosphorylationoccurring after cell adhesion to ECM, a transduction pathway


Crott�es et al.




triggered by the hERG/b1-integrin complex in acute leukemiasand colorectal cancer (7, 10).

We also provide evidence that Sig1R silencing decreases CMLand colorectal cancer cell motility not only in vitro, but also in vivoby using a zebrafish xenograft model (45). Interestingly, alteredmotility was paralleled by a reduced capacity for cells to reorga-nize their actin cytoskeleton, to promote the formation of actinspike, and to change their shape following contact with ECM(46).This result is in line with previous observations showing thathERG expression contributes to morphology, actin cytoskeletondynamics, and finally cell migration behavior (11). Together,these data reveal that Sig1R participates to cancer migrationpotency in response to ECM. Whether this function depends onthe formation of hERG signaling macrocomplex is confirmed bythe fact that hERG and AKT inhibitions mimicked Sig1R silencingin a nonadditive manner. In a good agreement with this hypoth-esis, the formation of the hERG/b1-integrin subunit complex inresponse to fibronectin increases cell migration ability by recruit-ing signaling proteins, such as focal adhesion kinases, PI3 kinases,and AKT in leukemias and colorectal cancer (7, 9, 10, 47).

Sig1R silencing impaired VEGF secretion in CML and colorectalcancer cells. We hypothesize that this effect is the consequence ofhERG regulation by Sig1R, as channel pharmacologic blockingalso reduced VEGF secretion. This idea is further confirmed by thefollowing observations: first, hERG/b1-integrin subunit complexcontrols VEGF secretion in AML, colorectal cancer, and glioblas-toma (7, 8, 10). Second, VEGF secretion is upregulated by AKTactivation in CML cells (40), a pathway triggered by hERG in CMLand colorectal cancer. Last, because VEGF has been shown tostimulate signaling pathways downstream to b1-integrin throughthe formation of a hERG/b1-integrin/Flt-1 complex in AML cells,it can be suggested that Sig1R participates to a VEGF-inducedautocrine-positive feedback further entertaining invasion (10). Ina pathologic context, VEGF secretion sustains angiogenesis (48)and cancer cell transendothelial migration (49), thus increasingblood stream invasion and dissemination to distant organs.We show that Sig1R increases angiogenesis in vivo, but alsostimulates transendothelial migration in vitro and lung extrava-sation in vivo. In other words, the presence of Sig1R in cancer cellslikely confers a proinvasive phenotype. Notably, hERG has beenlinked to enhanced invasion, angiogenesis, and bad prognosis inAML, CML, and colorectal cancer (10, 34, 50). Interestingly, weobserved that Sig1R is overexpressed in patients for these threecancers. We thus hypothesize that Sig1R, by promoting hERG ionchannel expression at the plasma membrane, increases invasivephenotype. Accordingly, AML patients with lower levels of Sig1RmRNA expression presented a better overall survival.

Recently, Sig1R has been implicated in the regulation of neu-ronal plasticity during neurologic disorders (stroke, addiction, orneurodegenerative diseases; refs. 13–16, 51). Indeed, the keymechanisms linking Sig1Rs to neuronal survival involve theirtranslocation from the MAM to plasma membrane, where theybind and modulate ion channels and receptors (14). Takentogether, our results suggest that the basic prosurvival function

of Sig1R is hijacked to adapt cancer cell behavior to signals fromthe microenvironment. In line with this new idea, we had previ-ously shown that Sig1R modulates volume-regulated chloridechannels in leukemia and small cell lung carcinoma cells toenhance resistance to apoptotic signals (52). Because thechaperoning activity of Sig1R can be regulated by exogenouscompounds (sigma ligands), we suggest that this protein may beused as a therapeutic target to specifically alter ion channel activityin cancerous tissues.

Disclosure of Potential Conflicts of InterestA. Paquet is Statistician at Laboratory Corporation of America. No potential

conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: D. Crott�es, A. Loubat, F. Borgese, O. SorianiDevelopment of methodology: D. Crott�es, R. Rapetti-Mauss, F. Alcaraz-Perez,H. Guizouarn, A. Loubat, M.L. Cayuela, F. BorgeseAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): D. Crott�es, R. Rapetti-Mauss, M. Tichet, G. Gariano,S. Martial, H. Guizouarn, A. Loubat, M. Presta, S. Tartare-Deckert, M.L. Cayuela,F. BorgeseAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): D. Crott�es, R. Rapetti-Mauss, F. Alcaraz-Perez,M. Tichet, G. Gariano, S. Martial, H. Guizouarn, A. Loubat, A. Popa, A. Paquet,M.L. Cayuela, F. Borgese, O. SorianiWriting, review, and/or revision of the manuscript:D. Crott�es, H. Guizouarn,A. Popa, F. Borgese, O. SorianiAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. Pellissier, S. Tartare-Deckert, P. Martin,O. SorianiStudy supervision: F. Borgese, O. SorianiOther (performed assays and interpreted results in the zebrafish model):F. Alcaraz-PerezOther (supervision zebrafish experiments): M. Presta

AcknowledgmentsThe microscopy was done in the Prism facility, "Plateforme PRISM – IBV,

CNRS UMR 7277, INSERM U1091-UNS." The help of Magali Mondin isacknowledged. The authors thank the Plateforme de G�enomique Fonctionnelle(IPMC, UNS, "France G�enomique" consortium; ANR-10-Infra-01), KevinLegrand for genomic dataset analysis, Ellen Van Obberghen-Schilling, Guil-laume Sandoz, and Gilles Pages for helpful discussions, and Ryan Arant forediting the article.

Grant SupportThis study was supported by UNS, CNRS, Ti'Toine Association and the

Ministero dell'Istruzione, Universit�a e Ricerca (FIRB Project 2011 "Infiamma-zione e cancro: approcci innovativi basati su nanotecnologie") and Associa-zione Italiana per la Ricerca sul Cancro (grant n� 14395) toM. Presta. D. Crott�esis a doctoral fellow with CNRS, R�egion PACA, and Fondation ARC. R. Rapetti-Mauss is a post-doctoral fellowof La Ligue contre leCancer. A. Popa is supportedby the Canceropole PACA.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received June 8, 2015; revised September 29, 2015; accepted October 27,2015; published OnlineFirst December 8, 2015.

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2016;76:607-618. Published OnlineFirst December 8, 2015.Cancer Res David Crottès, Raphael Rapetti-Mauss, Francisca Alcaraz-Perez, et al. Extracellular Matrix Stimulation to Drive Cancer Cell InvasivenessSIGMAR1 Regulates Membrane Electrical Activity in Response to

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